The Role And Mechanisms Of Protein Kinase D2 Promoting Epithelial Mesenchymal Transition(EMT) In Lung Adenocarcinoma

Lung cancer is the most common malignant tumor with the highest morbidity and mortality worldwide.According to the latest global cancer statistics,the incidence of lung cancer accounts for 11.6%of all malignant tumors,and the mortality rate accounts for 18.4%.Lung cancer can be divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC)according to the pathological type.Among them,NSCLC accounts for 85%of all lung cancer,and Lung adenocarcinoma is one of the pathological types in NSCLC.Despite that the traditional treatments for lung cancer,including surgery,radiotherapy,chemotherapy,etc.,have been greatly improved,the survival rate of lung cancer patients has not been greatly increased.In recent years,targeted therapies such as epidermal growth factor receptor inhibitors and immunotherapy such as PD-1 monoclonal antibody,as a new treatment,were gradually applied to the treatment of lung cancer,however,drug resistance and side effects has greatly limited the therapeutic effect on patients.Up to now,even in the developed United States,the five-year survival rate of lung cancer is only 19%.Therefore,it is urgent to find new and more effective treatment methods to improve the survival rate and quality of life of lung cancer patients.Protein kinase D(PKD)is a Ser/Thr protein kinase belonging to the Ca2+/calmodulin-dependent protein kinase(CAMK)family.PKD contains three highly conserved family members:PKD1,PKD2 and PKD3.The PKD2 gene encodes a protein of 878 amino acids weighted 105kDa.Like other protein kinases,PKD2 is activated by phosphorylation.The currently known phosphorylation site of PKD2 were Ser706,Ser710 in the catalytic domain and an autophosphorylation site at Ser876.In resting cells,PKD2 is mostly localized in the cytoplasm,and PKD2 can be phosphorylated or autophosphorylated when stimulated by hormones,growth factors,phorbol esters,G-protein coupled receptor agonists,etc.Then it can transfer to different structures of the cell,and act on different protein substrates to transmit signal information.With the molecular cloning of PKD2 reported,its role in various tumors has gradually revealed.Recent studies have shown that PKD2 is highly expressed in tumors including prostate cancer,pancreatic cancer,gastric cancer,breast cancer,colorectal cancer,leukemia,malignant glioma,and promote the development of them.In tumor cells,PKD2 can participate in the regulation of cell growth,cell proliferation,regulation of cell cycle and angiogenesis,regulation of tumor immune response,promotion of invasion and migration of tumor cells,promotion of cell survival and anti-apoptosis and other cell biological behaviors through PI3K/AKT/GSK3?/?-catenin,IKK?/NF?B,HIF-1?/VEGFA,MEK/ERK and other cellular pathways.Invasion and migration,as one of the common biological behaviors of malignant tumors,seriously affect the survival rate and quality of life of lung cancer patients.Due to the limitations of diagnosis and treatment,most patients have already had distant metastasis at the time of initial diagnosis.Epithelial mesenchymal transition(EMT)is one of the important behaviors of epithelial-derived tumors,which refers to the transition of the cell from epithelial phenotype to the interstitial phenotype.EMT can reduce the connection between epithelial cells,loose tissue structure,increase invasion and migration of tumor cells.Studies have shown that PKD2 can promote the invasion and migration of prostate cancer,malignant glioma,pancreatic cancer and other tumor cells,but its role in lung cancer,especially in lung adenocarcinoma,is still unclear.In this study,bioinformatics analysis was conducted and found that the high expression of PKD2 has a significant correlation with the occurrence and development of lung cancer and lymph node metastasis.Immunohistochemical staining analysis showed that high expression of PKD2 is a poor prognostic factor in patients with lung adenocarcinoma.Further experiments showed that PKD2 can induce EMT in lung adenocarcinoma cells through regulation the expression of p65,and the effect of PKD2 on the biological behavior of lung adenocarcinoma cells was confirmed by in vitro and in vivo experiments.The aim of our study is to elucidate the specific mechanism by which PKD2 promotes cancer progression and hope to provide a new target and theoretical basis for the treatment of lung adenocarcinoma.Part ? PKD2 was highly expressed and predicts poor prognosis in lung adenocarcinomaObjectives1.To investigate the expression and role of PKD2 in lung adenocarcinoma.2.To explore the prognostic role of PKD2 in patients with lung adenocarcinoma and its relationship with clinicopathological features.3.To explore the relationship between PKD2 and E-cadherin expression levels in lung adenocarcinoma tissues.Methods1.Bioinformatics analysis of the role of PKD2 in lung cancer:GSEA,Kaplan-Meier plotter and TCGA were used to analyze the relationship between PKD2 and the development,the prognostic role and lymph node metastasis in lung cancer.2.Twenty-seven pairs of lung adenocarcinoma tumor tissue paired with adjacent normal tissues were enrolled to verify the expression of PKD2 in tumor tissues by qRT-PCR and analyze its relationship with lymph node metastasis,staging and so on.3.Immunohistochemical staining of PKD2 was performed to analyze the prognosis value of PKD2 in lung adenocarcinoma patients and its relationship with clinicopathological features.4.Immunohistochemical staining of E-cadherin was performed to analyze the relationship between PKD2 and E-cadherin.PKD2 and E-cadherin were stained in the same tumor site in two tumor tissues,and the relationship between the two expressions was analyzed.Results1.The results of bioinformatics analysis showed that high PKD2 was significantly associated with the development,poor prognosis and lymph node metastasis in lung cancer.qRT-PCR confirmed that the expression level of PKD2 in lung adenocarcinoma was significantly higher than that in adjacent normal tissues.High expression of PKD2 was significantly correlated with lymph node metastasis and high stage(p<0.05).2.Immunohistochemical staining analysis showed that patients with higher PKD2 expression had lower overall survival(p=0.015)and progression-free survival(p=0.006),and it was an independent prognostic factor for patients with lung adenocarcinoma(HR= 2.150,95%Cl = 1.136-4.071,p = 0.01 9).3.PKD2 was negatively correlated with E-cadherin expression(p=0.021),low expression of E-cadherin was found in lung adenocarcinoma tissues with high expression of PKD2,and high expression of E-cadherin in tumors with low expression of PKD2.Conclusion1.PKD2 is highly expressed in lung adenocarcinoma tumor tissues and is associated with lymph node metastasis and advanced stage.2.High expression of PKD2 is significantly associated with poor prognosis and is an independent prognostic factor for patients with lung adenocarcinoma.3.PKD2 is negatively correlated with E-cadherin,suggesting that PKD2 may promote epithelial-mesenchymal transition in lung adenocarcinoma.Part ? The role and mechanisms of PKD2 promoting Epithelial mesenchymal transition(EMT)in lung adenocarcinomaObjectives1.To clarify the expression of PKD2 in various cell lines of lung cancer.2.To investigate the role of PKD2 in regulation of EMT and EMT-related transcription factors at the transcriptional level and protein level.3.To investigate the regulation of PKD2 expression on the expression of p65,a key factor in the NF?B pathway.4.The role of PKD2 in the biological behaviors such as proliferation,invasion and migration of lung adenocarcinoma cells was investigated in vitro and in vivo.Methods1.Western blot was used to detect the expression of PKD2 in each cell line.A549 and PC9 were selected for PKD2 phosphorylation under PMA stimulation.2.PKD2 plasmid was transfected into A549 and PC9 respectively.And the PKD2 knockdown cell line was constructed.The efficiency and the correlation between PKD2 and EMT markers at transcription and protein levels were verified by PCR and western blot.3.The regulation of PKD2 on p65 expression was verified by immunohisto-chemical staining,PCR and western blot.4.SRB staining,transwell,EDU assay,cell cycle and other experimental methods were used to verify the effect of PKD2 on the biological behavior of lung adenocarcinoma cells.5.Subcutaneous tumor formation model in nude mice was conducted to verify the effect of PKD2 on the proliferation of lung adenocarcinoma cells in vivo.Results1.High expression of PKD2 promoted the expression of EMT markers such as Vim,N-cadherin,MMP-9 and MMP-3 and EMT transcription factors such as snail1,snail2,zeb1 and twist,at mRNA and protein levels.In the PKD2 knock down cell lines,the EMT markers was reduced.And these changes in EMT indicators were not associated with PMA stimulation.2.Immunohistochemical staining showed that the expression of p65 in lung adenocarcinoma tissues with high expression of PKD2 was increased,while the low expression of PKD2 inhibited p65 expression.After transfection of PKD2,p65 mRNA and protein levels were increased in lung adenocarcinoma cell lines.After knocking down PKD2,p65 expression was decreased.3.When NF?B pathway was inhibited the effect of PKD2 inducing EMT was reduced.4.Cell biological behavior experiments showed that knocking down PKD2 inhibited the invasion,migration and proliferation of lung adenocarcinoma cells and caused arrested in G2/M phase.5.Subcutaneous tumor formation in nude mice confirmed that the growth rate of A549 tumors was significantly slowed down after knocking down of PKD2.Conclusions1.PKD2 significantly promotes EMT in lung adenocarcinoma cell lines.2.PKD2 can significantly promote the expression of p65.After inhibition of NF?B pathway,it can significantly inhibit the EMT induced by PKD2,indicating that PKD2 promotes EMT through NF?B/p65 pathway.3.PKD2 can promote the proliferation,invasion and migration of lung adenocarcinoma cell lines.It can promote tumor cell proliferation in vivo4.Studies have confirmed that PKD2 can induce EMT through the NF?B pathway to promote the development of lung adenocarcinoma.Targeting PKD2 is expected to become a new strategy in the treatment of lung adenocarcinoma.Part III Survival nomogram for patients with initially diagnosed metastatic non-small cell lung cancer:A SEER-Based StudyObjectivesThe object of this study was to construct an individualized predictive model to predict the overall survival(OS)of metastatic NSCLC(mNSCLC)patients.Methods1.We analyzed 11558 patients diagnosed with mNSCLC from 2010 to 2013 in the Surveillance,Epidemiology,and End Results(SEER)database.2.All patients were randomly divided into the training cohort(8670 cases)and validation cohort(2888 cases)by 75:25.Cox regression analyses were conducted to select independent prognostic factors.3.We evaluated the accuracy and the reliability of the model using concordance index(C-index),calibration curves and decision curve analyses(DCAs).We made a risk classification system based on the nomogram.Overall survival(OS)was compared between the two risk groups classified by total points in the model by Kaplan-Meier analysis.Results1.According to the results of Cox regression analysis,gender,age,race,marital status,primary site,pathological type,degree of differentiation,T stage,N stage,metastatic organs and chemotherapy were included in the model construction.2.The C-index was 0.732 and 0.730 for 1-and 2-year OS.3.The calibration plots presented good consistency between the nomogram prediction and actual observation.Great clinical applicability of the model was tested by DCA.The prognostic accuracy of the nomogram was better than AJCC TNM staging with evaluated integrated discrimination improvement(IDI).The low-risk group had significantly better OS than the high-risk group both in the training and the validation cohort.ConclusionsWe built a nomogram and a risk classification system based on the nomogram for predicting the OS of patients with initially diagnosed mNSCLC.The model can be a practical tool in treatment decision and individual counseling.